The goal of this project is to discover and optimize selective inhibitors of pyruvate kinase (PYK) from two protist parasites: Trypanosoma cruzi causes Chagas'disease throughout the Americas, including Texas and other southern regions of the United States, whereas two subspecies of Trypanosoma brucei cause sleeping sickness in areas of sub-Saharan Africa. Collectively parasites of the trypanosomatid family infect 30 million people worldwide (~500 million live in endemic areas), and account for ~130K deaths annually. Given their debilitating and prevalent nature, they impose a heavy medical, economic and social burden on entire populations primarily in the developing world. The problem is further exacerbated by the fact that existing treatments have progressed little in the past 50 years, and current drugs show high toxicity and poor efficacy. Glycolysis is essential in the infective stages of T. cruzi and T. brucei, and is therefore a promising drug target. Inhibitors of glycolytic enzymes such as PYK may thus serve as lead compounds for the development of new drugs. Furthermore, TbPYK has been validated as a drug target by RNAi, and detailed structural information is also available. In addition, trypanosomatid PYKs show significant differences from their human counterparts, especially with regard to allosteric properties. The proposed research has as specific aims: (1) To develop and validate qHTS assays mounted on the NCGC high-throughput screening platform for T. cruzi and T. brucei PYKs. (2) To explore a novel approach to HTS with assays containing the activator F16BP, thereby ensuring that the target enzyme will be in its activated conformation. The effector-bound (or R-state) conformation not only corresponds to the predominant conformation in vivo (for example, fructose bisphosphate levels are normally saturating in T. brucei cells), but is also a more rigid structure, with the consequence that inhibitors binding to R-state binding sites are likely to be entropically favoured. (3) To use these assays to screen the Molecular Library Small Molecule Repository (MLSMR) containing more than 300,000 small molecules. Unique structural features of trypanosomatid PYK (compared with the corresponding human enzymes) will be exploited for the discovery of selective inhibitors of these enzymes. (4) To confirm the potency of these compounds in a panel of secondary hit validation assays and to test their specificity in kinetic and selectivity assays, and to further improve the potency of the most promising molecules thus obtained by structure-based methods, analogue synthesis and medicinal chemical principles. (5) To determine the mode of action of the most promising molecules by enzyme assays, protein crystallography and biophysical measurements. (6) To test compounds displaying the highest potency for their ability to inhibit growth of cultured trypanosomatid cells representing pathogenic stages of the parasites, as well as for lack of toxicity toward cultured human cells. PUBLIC HEALTH RELEVANCE: Sleeping sickness in sub-Saharan Africa and Chagas'disease in the Americas (including the southern United States) cause severe public health and economic burdens on populations that are often already caught in a tragic cycle of poverty, poor nutrition and disease. Millions of people worldwide are infected by these potentially fatal diseases and hundreds of millions are at risk. Existing treatments have developed little in the past 50 years, and suffer from toxicity, inefficiency and resistance;the goal of this project is to develop lead drugs that will be suitable for entry into pre-clinical trials.